miRNAs Expression and Role of Dicer on Podocyte Injury in PAN Nephrosis Rats

Chunqing Li^1,2, Wei Sun^2,3*, Haochang Du¹, Dong Zhou³, Jihong Chen³, Lu Zhang³, Jiade Shao³

¹Nephrology Department of Wuxi No. 3 Hospital, Wuxi, China.
²Discipline of Chinese and Western Integrative Medicine, Nanjing University of Chinese Medicine, Nanjing, China.
³Nephrology Department of Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing, China.

DOI: 10.4236/cm.2015.61002                

Abstract

Objective: microRNAs (miRNAs) are regulatory RNAs that act as important players in diverse biologic and pathologic processes. Under circumstance as podocye-injury triggering proteinuria, which miRNAs are up-regulated or down-regulated? This experiment aims at detecting miRNAs changes in PAN nephrosis rats based on miRNA arrays and exploring the therapeutic targets of Leizhi capsule. Methods: Fifty male wistar rats were randomly divided into five groups, including control group, model group, leizhi capsule group, Tripterygium glucosides group, and valsartan group. PAN nephrosis models were made by jugular vein injection of PAN (100 mg/kg body weight, dissolve in physiological saline), while control group rats were made by jugular vein injection of physiological saline with equal volume. Other groups rats had been given medicines by irrigating stomach once a day for ten days. Blood and urine samples were collected, and renal tissues were processed after rats being euthanasised. The 24 h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed with light microscopy and transmission electron microscopy respectively. miRNA expression profile was detected by Exiqon miRNA Array. Real time RT-PCR analysis for mature miRNAs was used to validate differentially expressed miRNAs. Results: 1) In day 3 - 5, model rats had decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but which had no skin edema. Some rats died in serious ascites, the mortality is 3/10. 2) miRNA array detection shows 106 miRNAs up regulated and 62 miRNAs down regulated in PAN nephrosis rats. Fold change (model vs. control group) varies from 1.8 to 7.0. For leizhi capsule group and model sample, there are 90 miRNAs differentially expressed, with 65 miRNAs up and 25 miRNAs down. The most important finding in our research is the discovery of the specific miRNAs related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p), which have been validated by Real time RT-PCR analysis. 3) Compared with control sample, immune fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in PAN nephrosis rats. After treated with Leizhi Capsule, immune fluorescence intensity of the above molecules improved. Conclusion: 1) Characteristic miRNAs of PAN nephrosis were screening. Up-regulated miRNAs (rno-miR-23a, rno-miR-300-3p) may trigger podocyte injury and proteinuria, while down-regulated miRNAs (rno-miR-24, rno-miR-30c) may be protective factors by anti-apoptosis. 2) Dicer and these miRNAs (rno-miR-24, rno-miR-30c, rno-miR-23a) may be are probably key molecules therapeutic targets of Leizhi capsule.

Keywords

microRNAs, Podocyte Injury, Puromycin Aminonucleoside Nephrosis Model, Proteinuria, Leizhi Capsule

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Lee, R.C., Feinbaum, R.L. and Ambros, V. (1993) The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell, 75, 843-854. http://dx.doi.org/10.1016/0092-8674(93)90529-Y
[2]	Reinhart, B.J., Slack, F.J., Basson, M., et al. (2000) The 21-Nucleotide let-7 RNA Regulates Developmental Timing in Caenorhabditis elegans. Nature, 403, 901-906. http://dx.doi.org/10.1038/35002607
[3]	Muljo, S.A., Kanellopoulou, C. and Aravind, L. (2010) MicroRNA Targeting in Mammalian Genomes: Genes and Mechanisms. Wiley Interdisciplinary Reviews: Systems Biology and Medicine, 2, 148-161. http://dx.doi.org/10.1002/wsbm.53
[4]	Ohashi, T., Uchida, K., Asamiya, Y., et al. (2010) Phosphorylation Status of Nephrin in Human Membranous Nephropathy. Clinical and Experimental Nephrology, 14, 51-55. http://dx.doi.org/10.1007/s10157-009-0241-z
[5]	Welsh, G.I. and Saleem, M.A. (2010) Nephrin-Signature Molecule of the Glomerular Podocyte? Journal of Pathology, 220, 328-337.
[6]	Qin, X.S., Tsukaguchi, H., Shono, A., et al. (2009) Phosphorylation of Nephrin Triggers Its Internalization by Raft-Mediated Endocytosis. Journal of the American Society of Nephrology, 20, 2534-2545. http://dx.doi.org/10.1681/ASN.2009010011
[7]	Shi, S., Yu, L., Chiu, C., et al. (2008) Podocyte-Selective Deletion of Dicer Induces Proteinuria and Glomerulosclerosis. Journal of the American Society of Nephrology, 19, 2159-2169. http://dx.doi.org/10.1681/ASN.2008030312
[8]	Harvey, S.J., Jarad, G., Cunningham, J., et al. (2008) Podocyte-Specific Deletion of Dicer Alters Cytoskeletal Dynamics and Causes Glomerular Disease. Journal of the American Society of Nephrology, 19, 2150-2158. http://dx.doi.org/10.1681/ASN.2008020233
[9]	Ho, J., Ng, K.H., Rosen, S., et al. (2008) Podocyte-Specific Loss of Functional microRNAs Leads to Rapid Glomerular and Tubular Injury. Journal of the American Society of Nephrology, 19, 2069-2075. http://dx.doi.org/10.1681/ASN.2008020162
[10]	Liu, L.H., Zhu, C.F. and Ou, L.Z. (2005) Optimization and Evaluation of Puromycin Amioenucleoside Nephropathy Model in Rats. Fudan University Journal of Medical Sciences, 32, 488-492.
[11]	Chhabra, R., Dubey, R. and Saini, N. (2010) Cooperative and Individualistic Functions of the microRNAs in the miR-23a~27a~24-2 Cluster and Its Implication in Human Diseases. Molecular Cancer, 9, 232. http://dx.doi.org/10.1186/1476-4598-9-232
[12]	Chhabra, R., Adlakha, Y.K., Hariharan, M., et al. (2009) Upregulation of miR-23a-27a-24-2 Cluster Induces Caspase-Dependent and -Independent Apoptosis in Human Embryonic Kidney Cells. PLoS One, 4, e5848. http://dx.doi.org/10.1371/journal.pone.0005848
[13]	Griffiths-Jones, S. (2010) miRBase: microRNA Sequences and Annotation. Current Protocols in Bioinformatics, 12, 12-19.
[14]	Griffiths-Jones, S., Grocock, R.J., van Dongen, S., et al. (2006) miRBase: microRNA Sequences, Targets and Gene Nomenclature. Nucleic Acids Research, 34, D140-D144. http://dx.doi.org/10.1093/nar/gkj112